Compositions, comprising improved il-12 genetic constructs and vaccines, immunotherapeutics and methods of using the same

ABSTRACT

Nucleic acid molecules and compositions comprising: a nucleic acid sequence that encodes IL-12 p35 subunit or a functional fragment thereof and/or a nucleic acid sequence that encodes IL-12 p40 subunit or a functional fragment thereof, are disclosed. The nucleic acid molecules and compositions further comprising a nucleic acid sequence that encodes an immunogen are also disclosed. Method of modulating immune response and methods of inducing an immune response against an immunogen are disclosed. Therapeutic and prophylactic vaccination methods are also disclosed.

FIELD OF THE INVENTION

The present invention relates to improved genetic constructs that encodehuman IL-12 and nucleic acid molecules which comprise the same. Thepresent invention also relates to improved expression vectors, vaccinesand immunotherapeutics which include nucleotide sequences that encodehuman Il-12 and to methods of using the same.

BACKGROUND OF THE INVENTION

Immunotherapy refers to modulating a person's immune responses to imparta desirable therapeutic effect. Immunotherapeutics refer to thosecompositions which, when administered to an individual, modulate theindividual's immune system sufficient to ultimately decrease symptomswhich are associated with undesirable immune responses or to ultimatelyalleviate symptoms by increasing desirable immune responses. In somecases, immunotherapy is part of a vaccination protocol in which theindividual is administered a vaccine that exposes the individual to animmunogen against which the individual generates an immune response insuch cases, the immunotherapeutic increases the immune response and/orselectively enhances a portion of the immune response (such as thecellular arm or the humoral arm) which is desirable to treat or preventthe particular condition, infection or disease.

In designing vaccines, it has been recognized that vaccines that producethe target antigen in cells of the vaccinated individual are effectivein inducing the cellular arm of the immune system. Specifically, liveattenuated vaccines, recombinant vaccines which use avirulent vectorsand DNA vaccines each lead to the production of antigens in the cell ofthe vaccinated individual which results in induction of the cellular armof the immune system. On the other hand, killed or inactivated vaccines,and sub-unit vaccines which comprise only proteins do not induce goodcellular immune responses although they do induce an effective humoralresponse.

A cellular immune response is often necessary to provide protectionagainst pathogen infection and to provide effective immune-mediatedtherapy for treatment of pathogen infection, cancer or autoimmunediseases. Accordingly, vaccines that produce the target antigen in cellsof the vaccinated individual such as live attenuated vaccines,recombinant vaccines that use avirulent vectors and DNA vaccines areoften preferred.

There is a need for vaccine approaches that can induce strong T cell andB cell immunity in humans. Recent concerns over attenuation, vaccinemanufacturing complexity, serological interference, as was observed inthe HIV STEP trial, among a host of other issues serve to underscorethis important issue. In non-human primate models and in human clinicaltrials, simple plasmid DNA as a vaccine platform has not induced levelsof immunogenicity satisfactory for commercial development efforts to besupported. In head to head comparisons some naked plasmid-based vaccinesdid not induce either cellular or humoral responses comparable to thoseinduced by their viral vector counterparts, including the commonly usedadenovirus serotype 5 (Ad5) platform.

The development of DNA vaccine technology as a stand-alone method ofvaccination, as well as its utility in current prime-boost platforms,would benefit by the development of strategies to enhance its immunepotency. The manipulation of codon and RNA encoding sequences as well aschanges in leader sequences have been reported to enhance the expressionof plasmid-encoded immunogens. In addition, the creation of consensusimmunogens attempts to address the need for broad immunological coverageto account in part for viral diversity.

In addition, other strategies have been employed that focus on improvingthe physical delivery of DNA plasmids by improving formulations anddevice driven technologies. DNA vaccines delivered by electroporation(EP) have been reported to enhance antigen-specific interferon-γ (IFNγ)production following immunization of plasmid DNA in rhesus macaques.

The co-delivery of plasmid-encoded molecular adjuvants to augmentvaccine-induced responses is another important area of this specificinvestigation. One of the best-characterized molecular adjuvants innon-human primates is IL-12, a T_(H)1 polarizing cytokine that drivesCTL responses by providing the “third signal” needed for efficientactivation and antigen-specific expansion of naive CD8⁺ T cells. IL-12is a heterodimer which contains two subunits, p35 and p40. It has beenshown to be the most impressive immune enhancing cytokine, particularlyfor driving CD8 T cells when engineered as a DNA vaccine. In macaques,IL-12 has been shown to be an adjuvant that is highly potent forexpanding the cellular Immune potency of a DNA vaccine targetingmultiple antigens. In both macaques as well and in humans such a DNAvaccine adjuvant can significantly improve the immune responses inducedby a DNA vaccine.

U.S. Pat. No. 5,723,127, which is incorporated herein by reference,discloses IL-12 as a vaccine adjuvant. PCT application no.PCT/US1997/019502 and corresponding U.S. application Ser. No.08/956,865, which is incorporated herein by reference, discloses DNAvaccines and DNA constructs comprising IL-12 coding sequences.

There remains a need for improved vaccines and immunotherapeutics. Thereis a need for compositions and methods that produce enhanced immuneresponses. Likewise, while some immunotherapeutics are useful tomodulate immune response in a patient there remains a need for improvedimmunotherapeutic compositions and methods. There remains a need forimproved constructs which encode IL-12 and can be used as part of DNAvaccine strategies. There remains a need for improved constructs whichencode IL-12 and can be used as an immunotherapeutic. There remains aneed for improved constructs which encode IL-12 and can be used toachieve high levels of expression of IL-12.

SUMMARY OF THE INVENTION

Compositions are provided that comprises a nucleic acid sequence thatencodes IL-12 p35 subunit or a functional fragment thereof and a nucleicacid sequence that encodes IL-12 p40 subunit or a functional fragmentthereof. Nucleic acid sequences that encodes IL-12 p35 subunit may be atleast 98% homologous to SEQ ID NO:1 and encode a protein at least 98%homologous to SEQ ID NO:2. Nucleic acid sequences that encodesfunctional fragment of IL-12 p35 subunit may be fragments of a nucleicacid sequence that is at least 98% homologous to SEQ ID NO:1 and encodesa protein at least 98% homologous to a functional fragment of SEQ IDNO:2. Nucleic acid sequences that encodes IL-12 p40 subunit may be atleast 98% homologous to SEQ ID NO:3 and encode a protein at least 98%homologous to SEQ ID NO:4. Nucleic acid sequences that encodesfunctional fragment of IL-12 p40 subunit may be fragments of a nucleicacid sequence that is at least 98% homologous to SEQ ID NO:3 and encodesa protein at least 98% homologous to a functional fragment of SEQ IDNO:4. Compositions may further comprise a nucleic acid sequence thatencodes an immunogen.

Method of modulating immune response are also provided. The methodscomprise the step of administering to an individual, a composition thatcomprises a nucleic acid sequence that encodes IL-12 p35 subunit or afunctional fragment thereof and a nucleic acid sequence that encodesIL-12 p40 subunit or a functional fragment thereof.

Method of inducing an immune response against an immunogen are alsoprovided. The methods comprise the step of administering to anindividual, a composition that encodes IL-12 p35 subunit or a functionalfragment thereof and a nucleic acid sequence that encodes IL-12 p40subunit or a functional fragment thereof in combination with a nucleicacid sequence that encodes an immunogen in an amount. The methods ofinducing an immune response against an immunogen may be part of methodsof inducing a therapeutic immune response or methods of inducing aprophylactic immune response.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B shows a graph comparing expression levels of human IL-12in cells transfected with 2 μg HuIL12-opt or HuIL12-nonopt (FIG. 1A) or4 μg HuIL12-opt and HuIL12-nonopt (FIG. 1B).

FIG. 2 shows the enhanced PSA and PSMA-specific cellular immuneresponses in rhesus macaques.

FIG. 3 shows the enhanced HBV core and surface antigen-specific cellularimmune responses in rhesus macaques.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In one aspect of the invention, it is desired that the improved IL-12constructs provides for improved transcription and translation,including having one or more of the following: low GC content leadersequence to increase transcription; mRNA stability and codonoptimization; eliminating to the extent possible cis-acting sequencemotifs (i.e., internal TATA-boxes).

In some aspects of the invention, it is desired to incorporate theimproved IL-12 constructs into a vaccine regimen, either as part of thevaccine composition or as a separate composition delivered in acoordinated fashion with the vaccine in order to generate a broad immuneagainst vaccine immunogens. In some aspects of the invention, it isdesired to provide the improved IL-12 constructs as an immunotherapeuticwhich can be used to modulate immune responses in an individual. In someaspects of the invention, it is desired to provide the improved IL-12constructs in order to provide expression vectors which can be used toobtain high levels of IL-12 expression.

Higher potency IL-12 gene adjuvants are provided herein. These newadjuvants have several advantages over older IL-12 molecules. Anenhanced leader sequence that facilitates secretion of the molecules aswell as improves ribosome loading is provided, thus expanding the impactof these adjuvants and increasing expression. Significant changes to theRNA sequences further removes homology to native IL-12 sequences thuspreventing interference between the delivered adjuvant and the hostsystem, as well as lowering possible deleterious interactions betweenthe host IL-12 sequences and the gene delivered molecules. Furthermorethe higher potency of the new constructs lowers the dose requirementthus improving manufacturing as well as delivery issues associated withsuch adjuvants. Finally as these molecules have more bioactivity, theyimprove performance of the vaccine in vivo. Together these are importantnew tools for vaccine as well as immune therapy applications.

1. DEFINITIONS

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting. As used in thespecification and the appended claims, the singular forms “a,” “an” and“the” include plural referents unless the context clearly dictatesotherwise.

For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated.For example, for the range of 6-9, the numbers 7 and 8 are contemplatedin addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitlycontemplated.

a. Adjuvant “Adjuvant” as used herein may mean a molecule, including anucleic acid molecule that encodes a protein having immunomodulatingactivity, added to DNA plasmid vaccines or other vaccines to enhanceantigenicity of the one or more antigens encoded by the DNA plasmids orvaccines, and nucleic acid sequences that encode the adjuvant proteindescribed hereinafter.

b. Antibody “Antibody” may mean an antibody of classes IgG, IgM, IgA,IgD or IgE, or fragments, fragments or derivatives thereof, includingFab, F(ab′)2, Fd, and single chain antibodies, diabodies, bispecificantibodies, bifunctional antibodies and derivatives thereof. Theantibody may be an antibody isolated from the serum sample of mammal, apolyclonal antibody, affinity purified antibody, or mixtures thereofwhich exhibits sufficient binding specificity to a desired epitope or asequence derived therefrom.

c. Coding Sequence

“Coding sequence” or “encoding nucleic acid” as used herein may meanrefers to the nucleic acid (RNA or DNA molecule) that comprise anucleotide sequence which encodes a protein. The coding sequence mayfurther include initiation and termination signals operably linked toregulatory elements including a promoter and polyadenylation signalcapable of directing expression in the cells of an individual or mammalto whom the nucleic acid is administered.

d. Complement

“Complement” or “complementary” as used herein may mean a nucleic acidmay mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairingbetween nucleotides or nucleotide analogs of nucleic acid molecules.

e. Constant Current

“Constant current” as used herein to define a current that is receivedor experienced by a tissue, or cells defining said tissue, over theduration of an electrical pulse delivered to same tissue. The electricalpulse is delivered from the electroporation devices described herein.This current remains at a constant amperage in said tissue over the lifeof an electrical pulse because the electroporation device providedherein has a feedback element, preferably having instantaneous feedback.The feedback element can measure the resistance of the tissue (or cells)throughout the duration of the pulse and cause the electroporationdevice to alter its electrical energy output (e.g., increase voltage) socurrent in same tissue remains constant throughout the electrical pulse(on the order of microseconds), and from pulse to pulse. In someembodiments, the feedback element comprises a controller.

f. Current Feedback or Feedback

“Current feedback” or “feedback” as used herein may be usedinterchangeably and may mean the active response of the providedelectroporation devices, which comprises measuring the current in tissuebetween electrodes and altering the energy output delivered by the EPdevice accordingly in order to maintain the current at a constant level.This constant level is preset by a user prior to initiation of a pulsesequence or electrical treatment. The feedback may be accomplished bythe electroporation component, e.g., controller, of the electroporationdevice, as the electrical circuit therein is able to continuouslymonitor the current in tissue between electrodes and compare thatmonitored current (or current within tissue) to a preset current andcontinuously make energy-output adjustments to maintain the monitoredcurrent at preset levels. The feedback loop may be instantaneous as itis an analog closed-loop feedback.

g. Decentralized Current

“Decentralized current” as used herein may mean the pattern ofelectrical currents delivered from the various needle electrode arraysof the electroporation devices described herein, wherein the patternsminimize, or preferably eliminate, the occurrence of electroporationrelated heat stress on any area of tissue being electroporated.

h. Electroporation

“Electroporation,” “electro-permeabilization,” or “electro-kineticenhancement” (“EP”) as used interchangeably herein may refer to the useof a transmembrane electric field pulse to induce microscopic pathways(pores) in a bio-membrane; their presence allows biomolecules such asplasmids, oligonucleotides, siRNA, drugs, ions, and water to pass fromone side of the cellular membrane to the other.

i. Feedback Mechanism

“Feedback mechanism” as used herein may refer to a process performed byeither software or hardware (or firmware), which process receives andcompares the impedance of the desired tissue (before, during, and/orafter the delivery of pulse of energy) with a present value, preferablycurrent, and adjusts the pulse of energy delivered to achieve the presetvalue. A feedback mechanism may be performed by an analog closed loopcircuit.

j. Fragment

“Fragment” as used herein may mean a portion or a nucleic acid thatencodes a polypeptide capable of eliciting an immune response in amammal substantially similar to that of the non-fragment for Thefragments may be DNA fragments selected from fragments of SEQ ID NO:1,fragments of a nucleic acid sequence that is at least 98% homologous toSEQ ID NO:1 and encodes a function fragments of a protein that is atleast 98% homologous to SEQ ID NO:2; fragments of SEQ ID NO:3, andfragments of a nucleic acid sequence that is at least 98% homologous toSEQ ID NO:3 and encodes a function fragments of a protein that is atleast 98% homologous to SEQ ID NO:4.

The DNA fragments of SEQ ID NO:1, fragments of a nucleic acid sequencethat is at least 98% homologous to SEQ ID NO:1 and encodes a functionfragments of a protein that is at least 98% homologous to SEQ ID NO:2may encodes 50 or more amino acids in length, 55 or more, 60 or more, 65or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95or more, 100 or more, 105 or more, 110 or more, 115 or more, 120 ormore, 125 or more, 130 or more, 135 or more, 140 or more, 145 or more,150 or more, 155 or more, 160 or more, 165 or more, 170 or more, 175 ormore, 180 or more, 185 or more, 190 or more, 195 or more, 200 or more,205 or more, 210 or more in length or 215 or more of SEQ ID NO:2 or aprotein that is at least 98% homologous to SEQ ID NO:2 The DNA fragmentsof SEQ ID NO:1, fragments of a nucleic acid sequence that is at least98% homologous to SEQ ID NO:1 and encodes a function fragments of aprotein that is fewer than 53, fewer than 58, fewer than 63, fewer than68, fewer than 73, fewer than 78, fewer than 83, fewer than 88, fewerthan 93, fewer than 98, fewer than 103, fewer than 108, fewer than 113,fewer than 118, fewer than 123, fewer than 128, fewer than 133, fewerthan 138, fewer than 143, fewer than 148, fewer than 153, fewer than158, fewer than 163, fewer than 168, fewer than 173, fewer than 178,fewer than 183, fewer than 188, fewer than 193, fewer than 198, fewerthan 203, fewer than 208, fewer than 213 or fewer than 218 amino acidsin length of SEQ ID NO:2 or a protein that is at least 98% homologous toSEQ ID NO:2. In some embodiments, the fragments of a nucleic acidsequence that is at least 98% homologous to SEQ ID NO:1 encodesfunctional fragments of a protein that is at least 98% homologous to SEQID NO:2. In some embodiments, the fragments of a nucleic acid sequencethat is at least 98% homologous to SEQ ID NO:1 encodes functionalfragments of a protein that is at least 99% homologous to SEQ ID NO:2.In some embodiments, the fragments of a nucleic acid sequence that is atleast 98% homologous to SEQ ID NO:1 encodes functional fragments of SEQID NO:2. In some embodiments, the fragments of a nucleic acid sequencethat is at least 99% homologous to SEQ ID NO:1 encodes functionalfragments of a protein that is at least 98% homologous to SEQ ID NO:2.In some embodiments, the fragments of a nucleic acid sequence that is atleast 99% homologous to SEQ ID NO:1 encodes functional fragments of aprotein that is at least 99% homologous to SEQ ID NO:2. In someembodiments, the fragments of a nucleic acid sequence that is at least99% homologous to SEQ ID NO:1 encodes functional fragments of SEQ IDNO:2. In some embodiments, the fragments are fragments of SEQ ID NO:1that encode functional fragments of SEQ ID NO:2.

The DNA fragments of SEQ ID NO:3, fragments of a nucleic acid sequencethat is at least 98% homologous to SEQ ID NO:3 and encodes a functionfragments of a protein that is at least 98% homologous to SEQ ID NO:4may encodes 50 or more amino acids in length, 55 or more, 60 or more, 65or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95or more, 100 or more, 105 or more, 110 or more, 115 or more, 120 ormore, 125 or more, 130 or more, 135 or more, 140 or more, 145 or more,150 or more, 155 or more, 160 or more, 165 or more, 170 or more, 175 ormore, 180 or more, 185 or more, 190 or more, 195 or more, 200 or more,205 or more, 210 or more, 215 or more, 220 or more, 225 or more, 230 ormore, 235 or more, 240 or more, 245 or more, 250 or more, 255 or more,260 or more, 265 or more, 270 or more, 275 or more, 280 or more, 285 ormore, 290 or more, 295 or more, 300 or more, 305 or more, 310 or more,315 or more, 320 or more, or 325 or more amino acids of SEQ ID NO:4 orof a protein that is at least 98% homologous to SEQ ID NO:4 The DNAfragments of SEQ ID NO:3 and fragments of a nucleic acid sequence thatis at least 98% homologous to SEQ ID NO:3 may encode a functionfragments that is fewer than 53, fewer than 58, fewer than 63, fewerthan 68, fewer than 73, fewer than 78, fewer than 83, fewer than 88,fewer than 93, fewer than 98, fewer than 103, fewer than 108, fewer than113, fewer than 118, fewer than 123, fewer than 128, fewer than 133,fewer than 138, fewer than 143, fewer than 148, fewer than 153, fewerthan 158, fewer than 163, fewer than 168, fewer than 173, fewer than178, fewer than 183, fewer than 188, fewer than 193, fewer than 198,fewer than 203, fewer than 208, fewer than 213, fewer than 218, fewerthan 223, fewer than 228, fewer than 233, fewer than 238, fewer than243, fewer than 248, fewer than 253, fewer than 258, fewer than 263,fewer than 268, fewer than 273, fewer than 278, fewer than 283, fewerthan 288, fewer than 293, fewer than 298, fewer than 303, fewer than308, fewer than 313, fewer than 318 or fewer than 328 amino acids SEQ IDNO:4 or a protein that is at least 98% homologous to SEQ ID NO:4. Insome embodiments, the fragments of a nucleic acid sequence that is atleast 98% homologous to SEQ ID NO:3 encodes functional fragments of aprotein that is at least 98% homologous to SEQ ID NO:4. In someembodiments, the fragments of a nucleic acid sequence that is at least98% homologous to SEQ ID NO:3 encodes functional fragments of a proteinthat is at least 99% homologous to SEQ ID NO:4. In some embodiments, thefragments of a nucleic acid sequence that is at least 98% homologous toSEQ ID NO:3 encodes functional fragments of SEQ ID NO:4. In someembodiments, the fragments of a nucleic acid sequence that is at least99% homologous to SEQ ID NO:3 encodes functional fragments of a proteinthat is at least 98% homologous to SEQ ID NO:4. In some embodiments, thefragments of a nucleic acid sequence that is at least 99% homologous toSEQ ID NO:3 encodes functional fragments of a protein that is at least99% homologous to SEQ ID NO:4. In some embodiments, the fragments of anucleic acid sequence that is at least 99% homologous to SEQ ID NO:3encodes functional fragments of SEQ ID NO:4. In some embodiments, thefragments are fragments of SEQ ID NO:3 that encode functional fragmentsof SEQ ID NO:4.

DNA fragments may be free of coding sequences for IL-12 signal peptide.DNA fragments may comprise coding sequences for the immunoglobulinsignal peptide such as IgE or IgG signal peptide sequences. Thus forexample, DNA fragments that encode an IL-12 p35 subunit not encode aminoacids 1-22 of SEQ ID NO:2 and, in some such embodiments, may comprisessequences that encode an immunoglobulin signal peptide such as IgEsignal peptide sequence (SEQ ID NO:5) or IgG signal peptide sequence.

“Fragment” may also refer to polypeptide fragments capable offunctioning substantially substantially similar to that of the fulllength polypeptide. The fragment of IL-12 p35 may be a fragment of SEQID NO:2 or a fragment of a polypeptide that is at least 98% homologousto a fragment of SEQ ID NO:2. The fragment of IL-12 p35 may be afragment of a polypeptide that is at least 99% homologous to a fragmentof SEQ ID NO:2. The fragment of IL-12 p35 may be a fragment as describedabove. The fragment of IL-12 p40 may be a fragment of SEQ ID NO:4 or afragment of a polypeptide that is at least 98% homologous to a fragmentof SEQ ID NO:4. The fragment of IL-12 p40 may be a fragment of apolypeptide that is at least 99% homologous to a fragment of SEQ IDNO:4. The fragment of IL-12 p40 may be a fragment as described above.

A “functional fragment” is meant to refer to a fragment of an IL-12subunit that less than complete p35 and/or less than complete p40sequence, that, can function substantially similarly to full length p35or p40. Such substantially similar function includes interaction withother proteins, subunits and receptors in a substantially same manner asthe full length p35 or p40 and when delivered in a manner that allowsfor formation of a heterodimer results in substantially the same effectas the IL-12 p35/p40 heterodimer.

k. Genetic Construct

The term “genetic construct” as used herein refers to the DNA or RNAmolecules that comprise a nucleotide sequence which encodes one or bothIL-12 subunits or a target protein or another (non-IL-12)immunomodulating protein. The coding sequence includes initiation andtermination signals operably linked to regulatory elements including apromoter and polyadenylation signal capable of directing expression inthe cells of the individual to whom the nucleic acid molecule isadministered.

l. Hyperproliferative

As used herein, the term “hyperproliferative diseases” is meant to referto those diseases and disorders characterized by hyperproliferation ofcells and the term “hyperproliferative-associated protein” is meant torefer to proteins that are associated with a hyperproliferative disease.

m. Identical

“Identical” or “identity” as used herein in the context of two or morenucleic acids or polypeptide sequences, may mean that the sequences havea specified percentage of residues that are the same over a specifiedregion. The percentage may be calculated by optimally aligning the twosequences, comparing the two sequences over the specified region,determining the number of positions at which the identical residueoccurs in both sequences to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the specified region, and multiplying the result by 100 toyield the percentage of sequence identity. In cases where the twosequences are of different lengths or the alignment produces one or morestaggered ends and the specified region of comparison includes only asingle sequence, the residues of single sequence are included in thedenominator but not the numerator of the calculation. When comparing DNAand RNA, thymine (T) and uracil (U) may be considered equivalent.Identity may be performed manually or by using a computer sequencealgorithm such as BLAST or BLAST 2.0.

n. Impedance

“Impedance” as used herein may be used when discussing the feedbackmechanism and can be converted to a current value according to Ohm'slaw, thus enabling comparisons with the preset current.

o. Immune Response

“Immune response” as used herein may mean the activation of a host'simmune system, e.g., that of a mammal, in response to the introductionof one or more RSV consensus antigen via the provided DNA plasmidvaccines. The immune response can be in the form of a cellular orhumoral response, or both.

p. Intracellular Pathogen

“Intracellular pathogen” as used herein, is meant to refer to a virus orpathogenic organism that, at least part of its reproductive or lifecycle, exists within a host cell and therein produces or causes to beproduced, pathogen proteins.

q. Nucleic Acid

“Nucleic acid” or “oligonucleotide” or “polynucleotide” as used hereinmay mean at least two nucleotides covalently linked together. Thedepiction of a single strand also defines the sequence of thecomplementary strand. Thus, a nucleic acid also encompasses thecomplementary strand of a depicted single strand. Many variants of anucleic acid may be used for the same purpose as a given nucleic acid.Thus, a nucleic acid also encompasses substantially identical nucleicacids and complements thereof. A single strand provides a probe that mayhybridize to a target sequence under stringent hybridization conditions.Thus, a nucleic acid also encompasses a probe that hybridizes understringent hybridization conditions.

Nucleic acids may be single stranded or double stranded, or may containportions of both double stranded and single stranded sequence. Thenucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, wherethe nucleic acid may contain combinations of deoxyribo- andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosineand isoguanine Nucleic acids may be obtained by chemical synthesismethods or by recombinant methods.

r. Operably Linked

“Operably linked” as used herein when referring to a gene operablylinked to a promoter refers to the linkage of the two components suchthat expression of the gene is under the control of a promoter withwhich it is spatially connected. A promoter may be positioned 5′(upstream) or 3′ (downstream) of a gene under its control. The distancebetween the promoter and a gene may be approximately the same as thedistance between that promoter and the gene it controls in the gene fromwhich the promoter is derived. As is known in the art, variation in thisdistance may be accommodated without loss of promoter function. Whenreferring to a signal peptide operable linked to a protein, the termrefers to the protein having the signal peptide incorporated as part ofthe protein in a manner that it can function as a signal peptide. Whenreferring to coding sequence that encodes a signal peptide operablelinked to coding sequence that encodes a protein, the term refers to thecoding sequences arranged such that the translation of the codingsequence produces a protein having the signal peptide incorporated aspart of the protein in a manner that it can function as a signalpeptide.

s. Promoter

“Promoter” as used herein may mean a synthetic or naturally-derivedmolecule which is capable of conferring, activating or enhancingexpression of a nucleic acid in a cell. A promoter may comprise one ormore specific transcriptional regulatory sequences to further enhanceexpression and/or to alter the spatial expression and/or temporalexpression of same. A promoter may also comprise distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A promoter may bederived from sources including viral, bacterial, fungal, plants,insects, and animals. A promoter may regulate the expression of a genecomponent constitutively, or differentially with respect to cell, thetissue or organ in which expression occurs or, with respect to thedevelopmental stage at which expression occurs, or in response toexternal stimuli such as physiological stresses, pathogens, metal ions,or inducing agents. Representative examples of promoters include thebacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lacoperator-promoter, tac promoter, SV40 late promoter, SV40 earlypromoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40late promoter and the CMV IE promoter.

t. Stringent Hybridization Conditions

“Stringent hybridization conditions” as used herein may mean conditionsunder which a first nucleic acid sequence (e.g., probe) will hybridizeto a second nucleic acid sequence (e.g., target), such as in a complexmixture of nucleic acids. Stringent conditions are sequence-dependentand will be different in different circumstances. Stringent conditionsmay be selected to be about 5-10° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence at a defined ionic strength pH.The T_(m) may be the temperature (under defined ionic strength, pH, andnucleic concentration) at which 50% of the probes complementary to thetarget hybridize to the target sequence at equilibrium (as the targetsequences are present in excess, at T_(m), 50% of the probes areoccupied at equilibrium). Stringent conditions may be those in which thesalt concentration is less than about 1.0 M sodium ion, such as about0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes (e.g.,about 10-50 nucleotides) and at least about 60° C. for long probes(e.g., greater than about 50 nucleotides). Stringent conditions may alsobe achieved with the addition of destabilizing agents such as formamide.For selective or specific hybridization, a positive signal may be atleast 2 to 10 times background hybridization. Exemplary stringenthybridization conditions include the following: 50% formamide, 5×SSC,and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65°C., with wash in 0.2×SSC, and 0.1% SDS at 65° C.

u. Substantially Complementary

“Substantially complementary” as used herein may mean that a firstsequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or99% identical to the complement of a second sequence over a region of 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or morenucleotides or amino acids, or that the two sequences hybridize understringent hybridization conditions.

v. Substantially Identical

“Substantially identical” as used herein may mean that a first andsecond sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 98% or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or withrespect to nucleic acids, if the first sequence is substantiallycomplementary to the complement of the second sequence.

w. Target Protein

“Target protein” as used herein is meant to refer to peptides andprotein which are part of vaccines or which are encoded by geneconstructs of DNA vaccines that act as target proteins for an immuneresponse. The terms “target protein” and “immunogen” are usedinterchangeably and refer to a protein against which an immune responsecan be elicited. The target protein is an immunogenic protein thatshares at least an epitope with a protein from the pathogen orundesirable cell-type such as a cancer cell or a cell involved inautoimmune disease against which an immune response is desired. Theimmune response directed against the target protein will protect theindividual against and/or treat the individual for the specificinfection or disease with which the target protein is associated.

x. Variant

“Variant” used herein with respect to a nucleic acid may mean (i) aportion or fragment of a referenced nucleotide sequence; (ii) thecomplement of a referenced nucleotide sequence or portion thereof; (iii)a nucleic acid that is substantially identical to a referenced nucleicacid or the complement thereof; or (iv) a nucleic acid that hybridizesunder stringent conditions to the referenced nucleic acid, complementthereof, or a sequences substantially identical thereto.

“Variant” with respect to a peptide or polypeptide that differs in aminoacid sequence by the insertion, deletion, or conservative substitutionof amino acids, but retain at least one biological activity. Variant mayalso mean a protein with an amino acid sequence that is substantiallyidentical to a referenced protein with an amino acid sequence thatretains at least one biological activity. A conservative substitution ofan amino acid, i.e., replacing an amino acid with a different amino acidof similar properties (e.g., hydrophilicity, degree and distribution ofcharged regions) is recognized in the art as typically involving a minorchange. These minor changes can be identified, in part, by consideringthe hydropathic index of amino acids, as understood in the art. Kyte etal., J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an aminoacid is based on a consideration of its hydrophobicity and charge. It isknown in the art that amino acids of similar hydropathic indexes can besubstituted and still retain protein function. In one aspect, aminoacids having hydropathic indexes of ±2 are substituted. Thehydrophilicity of amino acids can also be used to reveal substitutionsthat would result in proteins retaining biological function. Aconsideration of the hydrophilicity of amino acids in the context of apeptide permits calculation of the greatest local average hydrophilicityof that peptide, a useful measure that has been reported to correlatewell with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101,incorporated fully herein by reference. Substitution of amino acidshaving similar hydrophilicity values can result in peptides retainingbiological activity, for example immunogenicity, as is understood in theart. Substitutions may be performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hydrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

y. Vector

“Vector” used herein may mean a nucleic acid sequence containing anorigin of replication. A vector may be a plasmid, bacteriophage,bacterial artificial chromosome or yeast artificial chromosome. A vectormay be a DNA or RNA vector. A vector may be either a self-replicatingextrachromosomal vector or a vector which integrates into a host genome.

2. IL-12

Provided herein is a synthetic, constructs which encode human IL-12 p35(the α subunit) and p40 (the β subunit). The human IL-12 p35 subunit(SEQ ID NO:2) is a 219 amino acid protein which includes a signalpeptide at amino acids 1-22 and a mature protein sequence at positions23-219. The human IL-12 p40 subunit (SEQ ID NO:4) is a 328 amino acidprotein which includes a signal peptide at amino acids 1-22 and a matureprotein sequence at positions 23-328. Amino acids 40-90 of the humanIL-12 p40 subunit are referred to as the immunoglobulin domain; aminoacids 125-217 of the human IL-12 p40 subunit are referred to as thecytokine interleukin-12 p40 C-terminus domain.

In some embodiments, the IL-12 p35 subunit is encoded by a constructcomprising a coding sequence on one plasmid and the IL-12 p40 subunit isencoded by a construct comprising a coding sequence on a differentplasmid. In some embodiments, the construct which comprises the IL-12p35 subunit coding sequence and the construct which comprises the IL-12p40 subunit coding sequence are on the same plasmid but each constructhas its own promoter. In some embodiments, the construct which comprisesthe IL-12 p35 subunit coding sequence and the construct which comprisesthe IL-12 p40 subunit coding sequence are on the same plasmid and underthe control of a single promoter and separated by an IRES sequence. Insome embodiments, the construct which comprises the IL-12 p35 subunitcoding sequence and the construct which comprises the IL-12 p40 subunitcoding sequence are on the same plasmid and under the control of asingle promoter and separated by a coding sequence for a proteolyticcleavage site. In some embodiments, the construct which comprises theIL-12 p35 subunit coding sequence and the construct which comprises theIL-12 p40 subunit coding sequence are on the same plasmid and under thecontrol of a single promoter and the subunit are separated by a linkerwhich allows them to be active as a single chain protein

HuIL12-opt sequences are optimized sequences that encode human IL-12subunits. The sequence have lower homology with the host genome tochange the RNA structure and avoid criptic regulation sequences. Thesequences provide improved mRNA stability and expression.

The HuIL12-opt sequence that is the coding sequence that encodes humanIL-12 p35 subunit is disclosed in SEQ ID NO:1. The HuIL12-opt sequencethat is the 219 amino acid IL-12 p35 subunit amino acid sequence encodedthereby is disclosed as SEQ ID NO. 2. Amino acids 1-22 correspond to thesignal peptide. Amino acids 23-219 correspond to the mature proteinregion.

The HuIL12-opt sequence that is the coding sequence that encodes humanIL-12 p40 subunit is disclosed as SEQ ID NO:3. The HuIL12-opt sequencethat is the 328 amino acid IL-12 p40 subunit amino acid sequence encodedthereby is disclosed as SEQ ID NO. 4. Amino acids 1-22 correspond to theIL-12 signal peptide and amino acids 23-328 make up the mature protein.Analogous sequences for Rhesus IL-12 are RhIL12-opt sequences which areoptimized sequences that encode rhesus IL-12 subunits.

In some embodiments, the IL-12 signal peptide of the IL-12 p35 or p40subunit or both may be replaced with a different signal peptide such asanother immunoglobulin signal peptide, for example IgG or IgE (SEQ IDNO:5). Coding sequences that encode the IL-12 signal peptide of theIL-12 p35 or p40 subunit or both may be replaced with coding sequencesthat encode a different signal peptide such as another immunoglobulinsignal peptide, for example IgG or IgE (that is coding sequences thatencode SEQ ID NO:5). In some embodiments, the IL-12 p35 signal peptidemay be replaced with a different signal peptide such as anotherimmunoglobulin signal peptide, for example IgG or IgE (SEQ ID NO:5).Functional fragments of SEQ ID NO. 2 may be free of the IL-12 p35 signalpeptide sequence. In some embodiments, coding sequence that encodes theIL-12 p35 signal peptide may be replaced with a coding sequence fordifferent signal peptide such as a coding sequence for anotherimmunoglobulin signal peptide, for example a coding sequence for thesignal peptide of IgG or IgE (i.e a coding sequence that encodes SEQ IDNO:5). Nucleic acid sequences that are fragments of SEQ ID NO:1 may befree of the coding sequence for IL-12 p35 signal peptide. Functionalfragments of SEQ ID NO. 4 may be free of the IL-12 p40 signal peptidesequence. In some embodiments, coding sequence that encodes the IL-12p40 signal peptide may be replaced with a coding sequence for differentsignal peptide such as a coding sequence for another immunoglobulinsignal peptide, for example a coding sequence for the signal peptide ofIgG or IgE (i.e a coding sequence that encodes SEQ ID NO:5). Nucleicacid sequences that are fragments of SEQ ID NO:3 may be free of thecoding sequence for IL-12 p40 signal peptide. In calculating homology toSEQ ID NO:1 or SEQ ID NO:3 in coding sequences that do not encode theIL-12 p35 signal peptide or IL-12 p40 signal peptide, respectively, thecalculation is base upon a comparison of SEQ ID NO:1 or SEQ ID NO:3excluding the portion of SEQ ID NO:1 that encode the IL-12 p35 signalpeptide or the portion of SEQ ID NO:3 that encodes the IL-12 p40 signalpeptide.

3. PLASMID

Provided herein is a vector that is capable of expressing the IL-12constructs in the cell of a mammal in a quantity effective to modulatean immune response in the mammal. Each vector may comprise heterologousnucleic acid encoding the one or both subunits. The vector may be aplasmid. The plasmid may be useful for transfecting cells with nucleicacid encoding Il-12, which the transformed host cell is cultured andmaintained under conditions wherein expression of the IL-12 takes place.

The plasmid may comprise a nucleic acid encoding one or more antigens.The plasmid may further comprise an initiation codon, which may beupstream of the coding sequence, and a stop codon, which may bedownstream of the coding sequence. The initiation and termination codonmay be in frame with the coding sequence.

The plasmid may also comprise a promoter that is operably linked to thecoding sequence The promoter operably linked to the coding sequence maybe a promoter from simian virus 40 (SV40), a mouse mammary tumor virus(MMTV) promoter, a human immunodeficiency virus (HIV) promoter such asthe bovine immunodeficiency virus (BIV) long terminal repeat (LTR)promoter, a Moloney virus promoter, an avian leukosis virus (ALV)promoter, a cytomegalovirus (CMV) promoter such as the CMV immediateearly promoter, Epstein Barr virus (EBV) promoter, or a Rous sarcomavirus (RSV) promoter. The promoter may also be a promoter from a humangene such as human actin, human myosin, human hemoglobin, human musclecreatine, or human metalothionein. The promoter may also be a tissuespecific promoter, such as a muscle or skin specific promoter, naturalor synthetic. Examples of such promoters are described in US patentapplication publication no. US20040175727, the contents of which areincorporated by reference herein in its entirety.

The plasmid may also comprise a polyadenylation signal, which may bedownstream of the coding sequence. The polyadenylation signal may be aSV40 polyadenylation signal, LTR polyadenylation signal, bovine growthhormone (bGH) polyadenylation signal, human growth hormone (hGH)polyadenylation signal, or human β-globin polyadenylation signal. TheSV40 polyadenylation signal may be a polyadenylation signal from a pCEP4plasmid (Invitrogen, San Diego, Calif.).

The plasmid may also comprise an enhancer upstream of the codingsequence. The enhancer may be human actin, human myosin, humanhemoglobin, human muscle creatine or a viral enhancer such as one fromCMV, FMDV, RSV or EBV. Polynucleotide function enhances are described inU.S. Pat. Nos. 5,593,972, 5,962,428, and WO94/016737, the contents ofeach are fully incorporated by reference in their entireties.

The plasmid may also comprise a mammalian origin of replication in orderto maintain the plasmid extrachromosomally and produce multiple copiesof the plasmid in a cell. The plasmid may be pVAX1, pCEP4 or pREP4 fromInvitrogen (San Diego, Calif.), which may comprise the Epstein Barrvirus origin of replication and nuclear antigen EBNA-1 coding region,which may produce high copy episomal replication without integration.The backbone of the plasmid may be pAV0242. The plasmid may be areplication defective adenovirus type 5 (Ad5) plasmid.

The plasmid may also comprise a regulatory sequence, which may be wellsuited for gene expression in a cell into which the plasmid isadministered. The coding sequence may comprise a codon that may allowmore efficient transcription of the coding sequence in the host cell.

The coding sequence may also comprise an Ig leader sequence. The leadersequence may be 5′ of the coding sequence. The consensus antigensencoded by this sequence may comprise an N-terminal Ig leader followedby a consensus antigen protein. The N-terminal Ig leader may be IgE orIgG.

The plasmid may be pSE420 (Invitrogen, San Diego, Calif.), which may beused for protein production in Escherichia coli (E. coli). The plasmidmay also be pYES2 (Invitrogen, San Diego, Calif.), which may be used forprotein production in Saccharomyces cerevisiae strains of yeast. Theplasmid may also be of the MAXBAC™ complete baculovirus expressionsystem (Invitrogen, San Diego, Calif.), which may be used for proteinproduction in insect cells. The plasmid may also be pcDNA I or pcDNA3(Invitrogen, San Diego, Calif.), which maybe used for protein productionin mammalian cells such as Chinese hamster ovary (CHO) cells.

4. VACCINE

According to some embodiments of the invention, the delivery of anucleic acid sequence that encodes IL-12 or functional fragmentsthereof, in combination with a nucleic acid sequence that encodes animmunogen to an individual enhances the immune response against theimmunogen. When the nucleic acid molecules that encode the immunogensand IL-12 are taken up by cells of the individual, the immunogen andIL-12 are expressed in cells and the proteins are thereby delivered tothe individual. Aspects of the invention provide methods of deliveringthe coding sequences of the immunogen and IL-12 on a single nucleic acidmolecule, methods of delivering the coding sequences of the immunogenand IL-12 on different nucleic acid molecules and methods of deliveringthe coding sequences of the proteins as part of recombinant vaccines andas part of attenuated vaccines.

According to some aspects of the present invention, compositions andmethods are provided which prophylactically and/or therapeuticallyimmunize an individual against a pathogen or abnormal, disease-relatedcells. The vaccine may be any type of vaccine such as, a live attenuatedvaccine, a recombinant vaccine or a nucleic acid or DNA vaccine. Bydelivering nucleic acid molecules that encode an immunogen and IL-12 orfunctional fragments thereof the immune response induced by the vaccinemay be modulated. The IL-12 constructs are particularly useful whendelivered in combination with a nucleic acid molecule that encodes animmunogen such as for example as part of a plasmid or the genome of arecombinant vector or attenuated pathogen or cell. The IL-12 constructsmay be used in vaccines prophylactically in order to induce a protectiveimmune response in an uninfected or disease free individual. The IL-12constructs are particularly useful when delivered to induce a protectiveimmune response in humans. The IL-12 constructs may be used in vaccinestherapeutically in order to induce a immune response in an infected ordiseased individual. The IL-12 constructs are particularly useful whendelivered to induce a therapeutic immune response in humans. In someembodiments, nucleic acid molecules comprising the IL-12 constructs aredelivered in a cell free composition. In some embodiments, nucleic acidmolecules comprising the IL-12 constructs are delivered in a compositionfree of cancer cells. In some embodiments, comprising the IL-12constructs are administered free of any other cytokine

Provided herein are vaccine capable of generating in a mammal an immuneresponse against pathogens, immunogens expressed on cells associatedwith disease and other immunogens against which an immune response isdesired. The vaccine may comprise each plasmid as discussed above. Thevaccine may comprise a plurality of the plasmids, or combinationsthereof. The vaccine may be provided to induce a therapeutic orprophylactic immune response.

Genetic constructs may comprise a nucleotide sequence that encodes atarget protein or an immunomodulating protein operably linked toregulatory elements needed for gene expression. According to theinvention, combinations of gene constructs that include one constructthat comprises an expressible form of the nucleotide sequence thatencodes a target protein and one construct that includes an expressibleform of the nucleotide sequence that encodes an immunomodulating proteinare provided. Delivery into a living cell of the DNA or RNA molecule(s)that include the combination of gene constructs results in theexpression of the DNA or RNA and production of the target protein andone or more immunomodulating proteins. An enhanced immune responseagainst the target protein results.

The present invention may be used to immunize an individual againstpathogens such as viruses, prokaryote and pathogenic eukaryoticorganisms such as unicellular pathogenic organisms and multicellularparasites. The present invention is particularly useful to immunize anindividual against those pathogens which infect cells and which are notencapsulated such as viruses, and prokaryote such as gonorrhea, listeriaand shigella. In addition, the present invention is also useful toimmunize an individual against protozoan pathogens that include a stagein the life cycle where they are intracellular pathogens. Table 1provides a listing of some of the viral families and genera for whichvaccines according to the present invention can be made. DNA constructsthat comprise DNA sequences that encode the peptides that comprise atleast an epitope identical or substantially similar to an epitopedisplayed on a pathogen antigen such as those antigens listed on thetables are useful in vaccines. Moreover, the present invention is alsouseful to immunize an individual against other pathogens includingprokaryotic and eukaryotic protozoan pathogens as well as multicellularparasites such as those listed on Table 2.

TABLE 1 Viruses Picornavirus Family Genera: Rhinoviruses: (Medical)responsible for −50% cases of the common cold. Etheroviruses: (Medical)includes polioviruses, coxsackieviruses, echoviruses, and humanenteroviruses such as hepatitis A virus. Apthoviruses: (Veterinary)these are the foot and mouth disease viruses. Target antigens: VP1, VP2,VP3, VP4, VPG Calcivirus Family Genera: Norwalk Group of Viruses:(Medical) these viruses are an important causative agent of epidemicgastroenteritis. Togavirus Family Genera: Alphaviruses: (Medical andVeterinary) examples include Sindbis virus, RossRiver virus andVenezuelan Eastern & Western Equine encephalitis viruses. Reovirus:(Medical) Rubella virus. Flariviridae Family Examples include: (Medical)dengue, yellow fever, Japanese encephalitis, St. Louis encephalitis andtick borne encephalitis viruses. West Nile virus (Genbank NC001563,AF533540, AF404757, AF404756, AF404755, AF404754, AF404753, AF481864,M12294, AF317203, AF196835, AF260969, AF260968, AF260967, AF206518 andAF202541) Representative Target antigens: E NS5 C Hepatitis C Virus:(Medical) these viruses are not placed in a family yet but are believedto be either a togavirus or a flavivirus. Most similarity is withtogavirus family. Coronavirus Family: (Medical and Veterinary)Infectious bronchitis virus (poultry) Porcine transmissiblegastroenteric virus (pig) Porcine hemagglutinating encephalomyelitisvirus (pig) Feline infectious peritonitis virus (cats) Feline entericcoronavirus (cat) Canine coronavirus (dog) SARS associated coronavirusThe human respiratory coronaviruses cause about 40% of cases of commoncold. EX. 224E, OC43 Note - coronaviruses may cause non-A, B or Chepatitis Target antigens: E1—also called M or matrix protein E2—alsocalled S or Spike protein E3—also called BE or hemagglutin-elteroseglycoprotein (not present in all coronaviruses) N—nucleocapsidRhabdovirus Family Genera: Vesiculovirus, Lyssavirus: (medical andveterinary) rabies Target antigen: G protein, N protein FiloviridaeFamily: (Medical) Hemorrhagic fever viruses such as Marburg and Ebolavirus Paramyxovirus Family: Genera: Paramyxovirus: (Medical andVeterinary) Mumps virus, New Castle disease virus (important pathogen inchickens) Morbillivirus: (Medical and Veterinary) Measles, caninedistemper Pneumovirus: (Medical and Veterinary) Respiratory syncytialvirus Orthomyxovirus Family (Medical) The Influenza virus BunyavirusFamily Genera: Bunyavirus: (Medical) California encephalitis, La CrossePhlebovirus: (Medical) Rift Valley Fever Hantavirus: Puremala is ahemahagin fever virus Nairvirus (Veterinary) Nairobi sheep disease Alsomany unassigned bungaviruses Arenavirus Family (Medical) LCM, Lassafever virus Reovirus Family Genera: Reovirus: a possible human pathogenRotavirus: acute gastroenteritis in children Orbiviruses: (Medical andVeterinary) Colorado Tick fever, Lebombo (humans) equine encephalosis,blue tongue Retroyirus Family Sub-Family: Oncorivirinal: (Veterinary)(Medical) feline leukemia virus, HTLVI and HTLVII Lentivirinal: (Medicaland Veterinary) HIV, feline immunodeficiency virus, equine infections,anemia virus Spumavirinal Papovavirus Family Sub-Family: Polyomaviruses:(Medical) BKU and JCU viruses Sub-Family: Papillomavirus: (Medical) manyviral types associated with cancers or malignant progression ofpapilloma. Adenovirus (Medical) EX AD7, ARD., O.B. - cause respiratorydisease - some adenoviruses such as 275 cause enteritis ParvovirusFamily (Veterinary) Feline parvovirus: causes feline enteritis Felinepanleucopeniavirus Canine parvovirus Porcine parvovirus HerpesvirusFamily Sub-Family: alphaherpesviridue Genera: Simplexvirus (Medical)HSVI (Genbank X14112, NC001806), HSVII (NC001798) Varicella zoster:(Medical Veterinary) Pseudorabies varicella zoster Sub-Familybetaherpesviridae Genera: Cytomegalovirus (Medical) HCMV MuromegalovirusSub-Family. Gammaherpesviridae Genera: Lymphocryptovirus (Medical) EBV -(Burkitt's lymphoma) Poxvirus Family Sub-Family: Chordopoxviridae(Medical - Veterinary) Genera: Variola (Smallpox) Vaccinia (Cowpox)Parapoxivirus - Veterinary Auipoxvirus - Veterinary CapripoxvirusLeporipoxvirus Suipoxviru's Sub-Family: Entemopoxviridue HepadnavirusFamily Hepatitis B virus Unclassified Hepatitis delta virus

TABLE 2 Bacterial pathogens Pathogenic gram-positive cocci include:pneumococcal; staphylococcal; and streptococcal. Pathogenicgram-negative cocci include: meningococcal; and gonococcal. Pathogenicenteric gram-negative bacilli include: enterobacteriaceae; pseudomonas,acinetobacteria and eikenella, melioidosis; salmonella; shigellosis;haemophilus; chancroid; brucellosis; tularemia; yersinia (pasteurella);streptobacillus mortiliformis and spirillum; listeria monocytogenes;erysipelothrix rhusiopathiae; diphtheria, cholera, anthrax; donovanosis(granuloma inguinale); and bartonellosis. Pathogenic anaerobic bacteriainclude: tetanus; botulism; other clostridia; tuberculosis; leprosy; andother mycobacteria. Pathogenic spirochetal diseases include: syphilis; -treponematoses: yaws, pinta and endemic syphilis; and leptospirosis.Other infections caused by higher pathogen bacteria and pathogenic fungiinclude: actinomycosis; nocardiosis; cryptococcosis, blastomycosis,histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis, andmucormycosis; sporotrichosis; paracoccidiodomycosis, petriellidiosis,torulopsosis, mycetoma, and chromomycosis; and dermatophytosis.Rickettsial infections include rickettsial and rickettsioses. Examplesof mycoplasma and chlamydial infections include: mycoplasma pneurnoniae;lymphogranuloma venereum; psittacosis; and perinatal chlamydialinfections. Pathogenic eukaryotes Pathogenic protozoans and helminthsand infections thereby include: amebiasis; malaria; leishmaniasis;trypanosomiasis; toxoplasmosis; pneumocystis carinii; babesiosis;giardiasis; trichinosis; filariasis; schistosomiasis; nematodes;trematodes or flukes; and cestode (tapeworm) infections.

In order to produce a genetic vaccine to protect against pathogeninfection, genetic material that encodes immunogenic proteins againstwhich a protective immune response can be mounted must be included in agenetic construct as the coding sequence for the target. Because DNA andRNA are both relatively small and can be produced relatively easily, thepresent invention provides the additional advantage of allowing forvaccination with multiple pathogen antigens. The genetic construct usedin the genetic vaccine can include genetic material that encodes manypathogen antigens. For example, several viral genes may be included in asingle construct thereby providing multiple targets.

Tables 1 and 2 include lists of some of the pathogenic agents andorganisms for which genetic vaccines can be prepared to protect anindividual from infection by them.

In some embodiments, vaccines comprise the optimized IL-12 incombination with one or more DNA vaccine constructs set forth in thefollowing patent documents which are each incorporated herein byreference. In some embodiments, vaccines comprise the optimized IL-12 incombination with (human immunodeficiency virus) an HIV vaccine, an(hepatitis C virus) HCV vaccine, a human papilloma virus (HPV) vaccine,an influenza vaccine or an hTERT-targeted cancer vaccines as disclosedin PCT application PCT/US07/74769 and corresponding U.S. patentapplication Ser. No. 12/375,518. In some embodiments, vaccines comprisethe optimized IL-12 in combination with an Influenza vaccines disclosedin PCT application PCT/US08/83281 and corresponding U.S. patentapplication Ser. No. 12/269,824 or PCT application PCT/US11/22642 andcorresponding U.S. patent application Ser. No. 12/694,238. In someembodiments, vaccines comprise the optimized IL-12 in combination withan HCV vaccines disclosed in PCT application PCT/US08/081,627 andcorresponding U.S. patent application Ser. No. 13/127,008. In someembodiments, vaccines comprise the optimized IL-12 in combination withan HPV vaccines disclosed in PCT application PCT/US10/21869 andcorresponding U.S. patent application Ser. No. 12/691,588 or U.S.provisional application Ser. No. 61/442,162. In some embodiments,vaccines comprise the optimized IL-12 in combination with an Smallpoxvaccines disclosed in PCT application PCT/US09/045,420 and correspondingU.S. patent application Ser. No. 12/473,634. In some embodiments,vaccines comprise the optimized IL-12 in combination with an Chikungunyavaccines disclosed in PCT application PCT/US09/039,656 and correspondingU.S. patent application Ser. No. 12/936,186. In some embodiments,vaccines comprise the optimized IL-12 in combination with an foot andmouth disease virus (FMDV) vaccines disclosed in PCT applicationPCT/US10/55187. In some embodiments, vaccines comprise the optimizedIL-12 in combination with an Malaria vaccines disclosed in U.S.provisional application Ser. No. 61/386,973. In some embodiments,vaccines comprise the optimized IL-12 in combination with an prostatecancer vaccines disclosed in U.S. provisional application Ser. No.61/413,176 or U.S. provisional application Ser. No. 61/417,817. In someembodiments, vaccines comprise the optimized IL-12 in combination withan human cytomegalovirus (CMV) vaccines disclosed in U.S. provisionalapplication Ser. No. 61/438,089. In some embodiments, vaccines comprisethe optimized IL-12 in combination with Methicillin-ResistantStaphylococcus aureus (MRSA) vaccines disclosed in U.S. ProvisionalApplication Ser. No. 61/569,727, filed on Dec. 12, 2011, entitled“PROTEINS COMPRISING MRSA PBP2A AND FRAGMENTS THEREOF, NUCLEIC ACIDSENCODING THE SAME, AND COMPOSITIONS AND THEIR USE TO PREVENT AND TREATMRSA INFECTIONS” and designated attorney docket number 133172.04000(X5709) and its corresponding PCT Application claiming priority to U.S.Provisional Application Ser. No. 61/569,727, filed on the same day asthe application filed herewith, each of which incorporate by referencein their entireties. All patents and patent applications disclosedherein are incorporated by reference in their entireties.

Another aspect of the present invention provides a method of conferringa protective immune response against hyperproliferating cells that arecharacteristic in hyperproliferative diseases and to a method oftreating individuals suffering from hyperproliferative diseases.Examples of hyperproliferative diseases include all forms of cancer andpsoriasis.

It has been discovered that introduction of a genetic construct thatincludes a nucleotide sequence which encodes an immunogenic“hyperproliferating cell”-associated protein into the cells of anindividual results in the production of those proteins in the vaccinatedcells of an individual. To immunize against hyperproliferative diseases,a genetic construct that includes a nucleotide sequence that encodes aprotein that is associated with a hyperproliferative disease isadministered to an individual.

In order for the hyperproliferative-associated protein to be aneffective immunogenic target, it must be a protein that is producedexclusively or at higher levels in hyperproliferative cells as comparedto normal cells. Target antigens include such proteins, fragmentsthereof and peptides; which comprise at least an epitope found on suchproteins. In some cases, a hyperproliferative-associated protein is theproduct of a mutation of a gene that encodes a protein. The mutated geneencodes a protein that is nearly identical to the normal protein exceptit has a slightly different amino acid sequence which results in adifferent epitope not found on the normal protein. Such target proteinsinclude those which are proteins encoded by oncogenes such as myb, myc,fyn, and the translocation gene bcr/abl, ras, src, P53, neu, trk andEGRF. In addition to oncogene products as target antigens, targetproteins for anti-cancer treatments and protective regimens includevariable regions of antibodies made by B cell lymphomas and variableregions of T cell receptors of T cell lymphomas which, in someembodiments, are also used target antigens for autoimmune disease. Othertumor-associated proteins can be used as target proteins such asproteins that are found at higher levels in tumor cells including theprotein recognized by monoclonal antibody 17-IA and folate bindingproteins or PSA.

While the present invention may be used to immunize an individualagainst one or more of several forms of cancer, the present invention isparticularly useful to prophylactically immunize an individual who ispredisposed to develop a particular cancer or who has had cancer and istherefore susceptible to a relapse. Developments in genetics andtechnology as well as epidemiology allow for the determination ofprobability and risk assessment for the development of cancer inindividual. Using genetic screening and/or family health histories, itis possible to predict the probability a particular individual has fordeveloping any one of several types of cancer.

Similarly, those individuals who have already developed cancer and whohave been treated to remove the cancer or are otherwise in remission areparticularly susceptible to relapse and reoccurrence. As part of atreatment regimen, such individuals can be immunized against the cancerthat they have been diagnosed as having had in order to combat arecurrence. Thus, once it is known that an individual has had a type ofcancer and is at risk of a relapse, they can be immunized in order toprepare their immune system to combat any future appearance of thecancer.

The present invention provides a method of treating individualssuffering from hyperproliferative diseases. In such methods, theintroduction of genetic constructs serves as an immunotherapeutic,directing and promoting the immune system of the individual to combathyperproliferative cells that produce the target protein. In treating orpreventing cancer, embodiments which are free of cells are particularlyuseful.

The present invention provides a method of treating individualssuffering from autoimmune diseases and disorders by conferring a broadbased protective immune response against targets that are associatedwith autoimmunity including cell receptors and cells which produce“self”-directed antibodies.

T cell mediated autoimmune diseases include Rheumatoid arthritis (RA),multiple sclerosis (MS), Sjogren's syndrome, sarcoidosis, insulindependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactivearthritis, ankylosing spondylitis, scleroderma, polymyositis,dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis,Crohn's disease and ulcerative colitis. Each of these diseases ischaracterized by T cell receptors that bind to endogenous antigens andinitiate the inflammatory cascade associated with autoimmune diseases.Vaccination against the variable region of the T cells would elicit animmune response including CTLs to eliminate those T cells.

In RA, several specific variable regions of T cell receptors (TCRs) thatare involved in the disease have been characterized. These TCRs includeVβ-3, Vβ-14, 20 Vβ-17 and Vα-17. Thus, vaccination with a DNA constructthat encodes at least one of these proteins will elicit an immuneresponse that will target T cells involved in RA. See: Howell, M. D., etal., 1991 Proc. Nat. Acad. Sci. USA 88:10921-10925; Piliard, X., et al,1991 Science 253:325-329; Williams, W. V., et al., 1992 J. Clin. Invest.90:326-333; each of which is incorporated herein by reference. In MS,several specific variable regions of TCRs that are involved in thedisease have been characterized. These TCRs include Vβ-7, and Vα-10.Thus, vaccination with a DNA construct that encodes at least one ofthese proteins will elicit an immune response that will target T cellsinvolved in MS. See: Wucherpfennig, K. W., et al., 1990 Science248:1016-1019; Oksenberg, J. R., et al, 1990 Nature 345:344-346; each ofwhich is incorporated herein by reference.

In scleroderma, several specific variable regions of TCRs that areinvolved in the disease have been characterized. These TCRs includeVβ-6, Vβ-8, V β-14 and Vα-16, Vα-3C, Vα-7, Vα-14, Vα-15, Vα-16, Vα-28and Vα-12. Thus, vaccination with a DNA construct that encodes at leastone of these proteins will elicit an immune response that will target Tcells involved in scleroderma.

In order to treat patients suffering from a T cell mediated autoimmunedisease, particularly those for which the variable region of the TCR hasyet to be characterized, a synovial biopsy can be performed. Samples ofthe T cells present can be taken and the variable region of those TCRsidentified using standard techniques. Genetic vaccines can be preparedusing this information.

B cell mediated autoimmune diseases include Lupus (SLE), Grave'sdisease, myasthenia gravis, autoimmune hemolytic anemia, autoimmunethrombocytopenia, asthma, cryoglobulinemia, primary biliary sclerosisand pernicious anemia. Each of these diseases is characterized byantibodies that bind to endogenous antigens and initiate theinflammatory cascade associated with autoimmune diseases. Vaccinationagainst the variable region of antibodies would elicit an immuneresponse including CTLs to eliminate those B cells that produce theantibody.

In order to treat patients suffering from a B cell mediated autoimmunedisease, the variable region of the antibodies involved in theautoimmune activity must be identified. A biopsy can be performed andsamples of the antibodies present at a site of inflammation can betaken. The variable region of those antibodies can be identified usingstandard techniques. Genetic vaccines can be prepared using thisinformation.

In the case of SLE, one antigen is believed to be DNA. Thus, in patientsto be immunized against SLE, their sera can be screened for anti-DNAantibodies and a vaccine can be prepared which includes DNA constructsthat encode the variable region of such anti-DNA antibodies found in thesera.

Common structural features among the variable regions of both TCRs andantibodies are well known. The DNA sequence encoding a particular TCR orantibody can generally be found following well known methods such asthose described in Kabat, et al 1987 Sequence of Proteins ofImmunological Interest U.S. Department of Health and Human Services,Bethesda Md., which is incorporated herein by reference. In addition, ageneral method for cloning functional variable regions from antibodiescan be found in Chaudhary, V. K., et al, 1990 Proc. Natl. Acad. Sci. USA87:1066, which is incorporated herein by reference.

In addition to using expressible forms of immunomodulating proteincoding sequences to improve genetic vaccines, the present inventionrelates to improved attenuated live vaccines and improved vaccines thatuse recombinant vectors to deliver foreign genes that encode antigens.Examples of attenuated live vaccines and those using recombinant vectorsto deliver foreign antigens are described in U.S. Pat. Nos. 4,722,848;5,017,487; 5,077,044; 5,110,587; 5,112,749; 5,174,993; 5,223,424;5,225,336; 5,240,703; 5,242,829; 5,294,441; 5,294,548; 5,310,668;5,387,744; 5,389,368; 5,424,065; 5,451,499; 5,453,364; 5,462,734;5,470,734; and 5,482,713, which are each incorporated herein byreference. Gene constructs are provided which include the nucleotidesequence of the IL-12 constructs or functional fragments thereof,wherein the nucleotide sequence is operably linked to regulatorysequences that can function in the vaccine to effect expression. Thegene constructs are incorporated in the attenuated live vaccines andrecombinant vaccines to produce improved vaccines according to theinvention.

The vaccine may further comprise a pharmaceutically acceptableexcipient. The pharmaceutically acceptable excipient may be functionalmolecules as vehicles, adjuvants, carriers, or diluents. Thepharmaceutically acceptable excipient may be a transfection facilitatingagent, which may include surface active agents, such asimmune-stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPSanalog including monophosphoryl lipid A, muramyl peptides, quinoneanalogs, vesicles such as squalene and squalene, hyaluronic acid,lipids, liposomes, calcium ions, viral proteins, polyanions,polycations, or nanoparticles, or other known transfection facilitatingagents.

The transfection facilitating agent is a polyanion, polycation,including poly-L-glutamate (LGS), or lipid. The transfectionfacilitating agent is poly-L-glutamate, and more preferably, thepoly-L-glutamate is present in the vaccine at a concentration less than6 mg/ml. The transfection facilitating agent may also include surfaceactive agents such as immune-stimulating complexes (ISCOMS), Freundsincomplete adjuvant, LPS analog including monophosphoryl lipid A,muramyl peptides, quinone analogs and vesicles such as squalene andsqualene, and hyaluronic acid may also be used administered inconjunction with the genetic construct. In some embodiments, the DNAplasmid vaccines may also include a transfection facilitating agent suchas lipids, liposomes, including lecithin liposomes or other liposomesknown in the art, as a DNA-liposome mixture (see for example WO9324640),calcium ions, viral proteins, polyanions, polycations, or nanoparticles,or other known transfection facilitating agents. Preferably, thetransfection facilitating agent is a polyanion, polycation, includingpoly-L-glutamate (LGS), or lipid. Concentration of the transfectionagent in the vaccine is less than 4 mg/ml, less than 2 mg/ml, less than1 mg/ml, less than 0.750 mg/ml, less than 0.500 mg/ml, less than 0.250mg/ml, less than 0.100 mg/ml, less than 0.050 mg/ml, or less than 0.010mg/ml.

The pharmaceutically acceptable excipient may be one or more additionaladjuvants. An adjuvant may be other genes that are expressed from thesame or from an alternative plasmid or are delivered as proteins incombination with the plasmid above in the vaccine. The one or moreadjuvants may be proteins and/or nucleic acid molecules that encodeproteins selected from the group consisting of: α-interferon (IFN-α),β-interferon (IFN-β), γ-interferon, platelet derived growth factor(PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous Tcell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine(TECK), mucosae-associated epithelial chemokine (MEC), IL-15 includingIL-15 having the signal sequence or coding sequence that encodes thesignal sequence deleted and optionally including a different signalpeptide such as that from IgE or coding sequence that encodes adifference signal peptide such as that from IgE, IL-28, MHC, CD80, CD86,IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, MCP-1, MIP-1α, MIP-1β, IL-8,L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1,VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF,G-CSF, mutant forms of IL-18, CD40, CD40L, vascular growth factor,fibroblast growth factor, IL-7, nerve growth factor, vascularendothelial growth factor, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1,DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2,DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88,IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1, JNK, interferon responsegenes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4,RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B,NKG2C, NKG2E, NKG2F, TAP1, TAP2 and functional fragments thereof or acombination thereof. In some embodiments additional adjuvant may be oneor more proteins and/or nucleic acid molecules that encode proteinsselected from the group consisting of: IL-15, IL-28, CTACK, TECK, MEC orRANTES. Examples of IL-15 constructs and sequences are disclosed in PCTapplication no. PCT/US04/18962 and corresponding U.S. application Ser.No. 10/560,650, and in PCT application no. PCT/US07/00886 andcorresponding U.S. application Ser. No. 12/160,766, and in PCTapplication no. PCT/US10/048,827. Examples of IL-28 constructs andsequences are disclosed in PCT application no. PCT/US09/039,648 andcorresponding U.S. application Ser. No. 12/936,192. Examples of RANTESand other constructs and sequences are disclosed in PCT application no.PCT/US1999/004332 and corresponding U.S. application Ser. No. and09/622,452. Other examples of RANTES constructs and sequences aredisclosed in PCT application no. PCT/US11/024,098. Examples of RANTESand other constructs and sequences are disclosed in PCT application no.PCT/US1999/004332 and corresponding U.S. application Ser. No.09/622,452. Other examples of RANTES constructs and sequences aredisclosed in PCT application no. PCT/US11/024,098. Examples ofchemokines CTACK, TECK and MEC constructs and sequences are disclosed inPCT application no. PCT/US2005/042231 and corresponding U.S. applicationSer. No. 11/719,646. Examples of OX40 and other immunomodulators aredisclosed in U.S. application Ser. No. 10/560,653. Examples of DR5 andother immunomodulators are disclosed in U.S. application Ser. No.09/622,452.

The vaccine may further comprise a genetic vaccine facilitator agent asdescribed in U.S. Ser. No. 021,579 filed Apr. 1, 1994, which is fullyincorporated by reference.

The vaccine may comprise the consensus antigens and plasmids atquantities of from about 1 nanogram to 100 milligrams; about 1 microgramto about 10 milligrams; or preferably about 0.1 microgram to about 10milligrams; or more preferably about 1 milligram to about 2 milligram.In some preferred embodiments, pharmaceutical compositions according tothe present invention comprise about 5 nanogram to about 1000 microgramsof DNA. In some preferred embodiments, the pharmaceutical compositionscontain about 10 nanograms to about 800 micrograms of DNA. In somepreferred embodiments, the pharmaceutical compositions contain about 0.1to about 500 micrograms of DNA. In some preferred embodiments, thepharmaceutical compositions contain about 1 to about 350 micrograms ofDNA. In some preferred embodiments, the pharmaceutical compositionscontain about 25 to about 250 micrograms, from about 100 to about 200microgram, from about 1 nanogram to 100 milligrams; from about 1microgram to about 10 milligrams; from about 0.1 microgram to about 10milligrams; from about 1 milligram to about 2 milligram, from about 5nanogram to about 1000 micrograms, from about 10 nanograms to about 800micrograms, from about 0.1 to about 500 micrograms, from about 1 toabout 350 micrograms, from about 25 to about 250 micrograms, from about100 to about 200 microgram of the consensus antigen or plasmid thereof.

The vaccine may be formulated according to the mode of administration tobe used. An injectable vaccine pharmaceutical composition may besterile, pyrogen free and particulate free. An isotonic formulation orsolution may be used. Additives for isotonicity may include sodiumchloride, dextrose, mannitol, sorbitol, and lactose. The vaccine maycomprise a vasoconstriction agent. The isotonic solutions may includephosphate buffered saline. Vaccine may further comprise stabilizersincluding gelatin and albumin. The stabilizing may allow the formulationto be stable at room or ambient temperature for extended periods of timesuch as LGS or polycations or polyanions to the vaccine formulation.

5. METHODS OF DELIVERY THE VACCINE

Provided herein is a method for delivering a vaccine including the IL-12constructs to produce immune responses effective against the vaccineimmunogens. The method of delivering the vaccine or vaccination may beprovided to induce a therapeutic and prophylactic immune response. Thevaccination process may generate in the mammal an immune responseagainst immunogens. The vaccine may be delivered to an individual tomodulate the activity of the mammal's immune system and enhance theimmune response. The delivery of the vaccine may be the transfection ofsequences encoding the immunogen and the IL-12 constructs on one or morenucleic acid molecules. The coding sequences are expressed in cells anddelivered to the surface of the cell upon which the immune systemrecognized and induces a cellular, humoral, or cellular and humoralresponse. The delivery of the vaccine may be use to induce or elicit andimmune response in mammals against the immunogen by administering to themammals the vaccine as discussed above. The inclusion of the IL-12constructs results in a more effective immune response.

Upon delivery of the vaccine and plasmid into the cells of the mammal,the transfected cells will express and secrete immunogens and IL-12encoded by the plasmids injected from the vaccine. These immunogens willbe recognized as foreign by the immune system and antibodies will bemade against them. These antibodies will be maintained by the immunesystem and allow for an effective response to subsequent infections. Thepresence of the IL-12 encoded by the IL-12 constructs results in agreater immune response.

The vaccine may be administered to a mammal to elicit an immune responsein a mammal. The mammal may be human, primate, non-human primate, cow,cattle, sheep, goat, antelope, bison, water buffalo, bison, bovids,deer, hedgehogs, elephants, llama, alpaca, mice, rats, and chicken.

a. Combination Treatments

The IL-12 construct may be administered in combination with otherproteins or genes encoding one or more of α-interferon, γ-interferon,platelet derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermalgrowth factor (EGF), cutaneous T cell-attracting chemokine (CTACK),epithelial thymus-expressed chemokine (TECK), mucosae-associatedepithelial chemokine (MEC), IL-15 (including IL-15 having the signalsequence deleted and optionally including the signal peptide from IgE),MHC, CD80, CD86, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-18, IL-28,MCP-1, MIP-1α, MIP-1β, IL-8, RANTES, L-selectin, P-selectin, E-selectin,CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1,ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18,CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7,nerve growth factor, vascular endothelial growth factor, Fas, TNFreceptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF,DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1,Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K,SAP-1, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec,TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND,NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 andfunctional fragments thereof or combinations thereof.

The vaccine may be administered by different routes including orally,parenterally, sublingually, transdermally, rectally, transmucosally,topically, via inhalation, via buccal administration, intrapleurally,intravenous, intraarterial, intraperitoneal, subcutaneous,intramuscular, intranasal intrathecal, and intraarticular orcombinations thereof. For veterinary use, the composition may beadministered as a suitably acceptable formulation in accordance withnormal veterinary practice. The veterinarian can readily determine thedosing regimen and route of administration that is most appropriate fora particular animal. The vaccine may be administered by traditionalsyringes, needleless injection devices, “microprojectile bombardmentgone guns”, or other physical methods such as electroporation (“EP”),“hydrodynamic method”, or ultrasound.

The plasmid of the vaccine may be delivered to the mammal by severalwell known technologies including DNA injection (also referred to as DNAvaccination) with and without in vivo electroporation, liposomemediated, nanoparticle facilitated, recombinant vectors such asrecombinant adenovirus, recombinant adenovirus associated virus andrecombinant vaccinia. The consensus antigen may be delivered via DNAinjection and along with in vivo electroporation.

b. Electroporation

Administration of the vaccine via electroporation of the plasmids of thevaccine may be accomplished using electroporation devices that can beconfigured to deliver to a desired tissue of a mammal a pulse of energyeffective to cause reversible pores to form in cell membranes, andpreferable the pulse of energy is a constant current similar to a presetcurrent input by a user. The electroporation device may comprise anelectroporation component and an electrode assembly or handle assembly.The electroporation component may include and incorporate one or more ofthe various elements of the electroporation devices, including:controller, current waveform generator, impedance tester, waveformlogger, input element, status reporting element, communication port,memory component, power source, and power switch. The electroporationmay be accomplished using an in vivo electroporation device, for exampleCELLECTRA EP system (Inovio Pharmaceuticals, Blue Bell, Pa.) or Elgenelectroporator (Genetronics, San Diego, Calif.) to facilitatetransfection of cells by the plasmid.

The electroporation component may function as one element of theelectroporation devices, and the other elements are separate elements(or components) in communication with the electroporation component. Theelectroporation component may function as more than one element of theelectroporation devices, which may be in communication with still otherelements of the electroporation devices separate from theelectroporation component. The elements of the electroporation devicesexisting as parts of one electromechanical or mechanical device may notlimited as the elements can function as one device or as separateelements in communication with one another. The electroporationcomponent may be capable of delivering the pulse of energy that producesthe constant current in the desired tissue, and includes a feedbackmechanism. The electrode assembly may include an electrode array havinga plurality of electrodes in a spatial arrangement, wherein theelectrode assembly receives the pulse of energy from the electroporationcomponent and delivers same to the desired tissue through theelectrodes. At least one of the plurality of electrodes is neutralduring delivery of the pulse of energy and measures impedance in thedesired tissue and communicates the impedance to the electroporationcomponent. The feedback mechanism may receive the measured impedance andcan adjust the pulse of energy delivered by the electroporationcomponent to maintain the constant current.

A plurality of electrodes may deliver the pulse of energy in adecentralized pattern. The plurality of electrodes may deliver the pulseof energy in the decentralized pattern through the control of theelectrodes under a programmed sequence, and the programmed sequence isinput by a user to the electroporation component. The programmedsequence may comprise a plurality of pulses delivered in sequence,wherein each pulse of the plurality of pulses is delivered by at leasttwo active electrodes with one neutral electrode that measuresimpedance, and wherein a subsequent pulse of the plurality of pulses isdelivered by a different one of at least two active electrodes with oneneutral electrode that measures impedance.

The feedback mechanism may be performed by either hardware or software.The feedback mechanism may be performed by an analog closed-loopcircuit. The feedback occurs every 50 μs, 20 μs, 10 μs or 1 μs, but ispreferably a real-time feedback or instantaneous (i.e., substantiallyinstantaneous as determined by available techniques for determiningresponse time). The neutral electrode may measure the impedance in thedesired tissue and communicates the impedance to the feedback mechanism,and the feedback mechanism responds to the impedance and adjusts thepulse of energy to maintain the constant current at a value similar tothe preset current. The feedback mechanism may maintain the constantcurrent continuously and instantaneously during the delivery of thepulse of energy.

Examples of electroporation devices and electroporation methods that mayfacilitate delivery of the DNA vaccines of the present invention,include those described in U.S. Pat. No. 7,245,963 by Draghia-Akli, etal., U.S. Patent Pub. 2005/0052630 submitted by Smith, et al., thecontents of which are hereby incorporated by reference in theirentirety. Other electroporation devices and electroporation methods thatmay be used for facilitating delivery of the DNA vaccines include thoseprovided in co-pending and co-owned U.S. patent application Ser. No.11/874,072, filed Oct. 17, 2007, which claims the benefit under 35 USC119(e) to U.S. Provisional Application Ser. Nos. 60/852,149, filed Oct.17, 2006, and 60/978,982, filed Oct. 10, 2007, all of which are herebyincorporated in their entirety.

U.S. Pat. No. 7,245,963 by Draghia-Akli, et al. describes modularelectrode systems and their use for facilitating the introduction of abiomolecule into cells of a selected tissue in a body or plant. Themodular electrode systems may comprise a plurality of needle electrodes;a hypodermic needle; an electrical connector that provides a conductivelink from a programmable constant-current pulse controller to theplurality of needle electrodes; and a power source. An operator cangrasp the plurality of needle electrodes that are mounted on a supportstructure and firmly insert them into the selected tissue in a body orplant. The biomolecules are then delivered via the hypodermic needleinto the selected tissue. The programmable constant-current pulsecontroller is activated and constant-current electrical pulse is appliedto the plurality of needle electrodes. The applied constant-currentelectrical pulse facilitates the introduction of the biomolecule intothe cell between the plurality of electrodes. The entire content of U.S.Pat. No. 7,245,963 is hereby incorporated by reference.

U.S. Patent Pub. 2005/0052630 submitted by Smith, et al. describes anelectroporation device which may be used to effectively facilitate theintroduction of a biomolecule into cells of a selected tissue in a bodyor plant. The electroporation device comprises an electro-kinetic device(“EKD device”) whose operation is specified by software or firmware. TheEKD device produces a series of programmable constant-current pulsepatterns between electrodes in an array based on user control and inputof the pulse parameters, and allows the storage and acquisition ofcurrent waveform data. The electroporation device also comprises areplaceable electrode disk having an array of needle electrodes, acentral injection channel for an injection needle, and a removable guidedisk. The entire content of U.S. Patent Pub. 2005/0052630 is herebyincorporated by reference.

The electrode arrays and methods described in U.S. Pat. No. 7,245,963and U.S. Patent Pub. 2005/0052630 may be adapted for deep penetrationinto not only tissues such as muscle, but also other tissues or organs.Because of the configuration of the electrode array, the injectionneedle (to deliver the biomolecule of choice) is also insertedcompletely into the target organ, and the injection is administeredperpendicular to the target issue, in the area that is pre-delineated bythe electrodes The electrodes described in U.S. Pat. No. 7,245,963 andU.S. Patent Pub. 2005/005263 are preferably 20 mm long and 21 gauge.

Additionally, contemplated in some embodiments that incorporateelectroporation devices and uses thereof, there are electroporationdevices that are those described in the following patents: U.S. Pat. No.5,273,525 issued Dec. 28, 1993, U.S. Pat. No. 6,110,161 issued Aug. 29,2000, U.S. Pat. No. 6,261,281 issued Jul. 17, 2001, and U.S. Pat. No.6,958,060 issued Oct. 25, 2005, and U.S. Pat. No. 6,939,862 issued Sep.6, 2005. Furthermore, patents covering subject matter provided in U.S.Pat. No. 6,697,669 issued Feb. 24, 2004, which concerns delivery of DNAusing any of a variety of devices, and U.S. Pat. No. 7,328,064 issuedFeb. 5, 2008, drawn to method of injecting DNA are contemplated herein.The above-patents are incorporated by reference in their entirety.

c. Method of Preparing DNA Plasmids

Provided herein is methods for preparing the DNA plasmids that comprisethe DNA constructs and vaccines discussed herein. The DNA plasmids,after the final subcloning step into the mammalian expression plasmid,can be used to inoculate a cell culture in a large scale fermentationtank, using known methods in the art.

The DNA plasmids for use with the EP devices of the present inventioncan be formulated or manufactured using a combination of known devicesand techniques, but preferably they are manufactured using an optimizedplasmid manufacturing technique that is described in a licensed,co-pending U.S. provisional application U.S. Ser. No. 60/939,792, whichwas filed on May 23, 2007. In some examples, the DNA plasmids used inthese studies can be formulated at concentrations greater than or equalto 10 mg/mL. The manufacturing techniques also include or incorporatevarious devices and protocols that are commonly known to those ofordinary skill in the art, in addition to those described in U.S. Ser.No. 60/939,792, including those described in a licensed patent, U.S.Pat. No. 7,238,522, which issued on Jul. 3, 2007. The above-referencedapplication and patent, U.S. Ser. No. 60/939,792 and U.S. Pat. No.7,238,522, respectively, are hereby incorporated in their entirety.

6. IMMUNOMODULATING COMPOSITIONS AND METHODS

In some embodiments, the nucleic acid sequences that encode the IL-12subunits are delivered without the addition of nucleic acid sequencesthat encode an immunogen. In such methods, the nucleic acid sequencesthat encode the IL-12 subunits are used as immunotherapeutics which,when expressed to produce functional IL-12, impart a desiredimmunomodulatory effect on the individual. The nucleic acid sequencesthat encode the IL-12 subunits are provided and delivered as describedabove except for the exclusion of nucleic acid sequences that encode animmunogen. In such methods, the nucleic acid sequences that encode theIL-12 subunits may used as immunotherapeutics alone or in combinationwith other immunomodulatory proteins such as those described above inthe section entitled combination treatments.

EXAMPLES

The present invention is further illustrated in the following Examples.It should be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, various modifications of the invention in addition tothose shown and described herein will be apparent to those skilled inthe art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims

Example 1 Comparing Expression Levels of phuIL12-Opt with phuIL12-Nonopt

Comparison of the expression levels of phuIL12-opt with phuIL12-nonoptwas performed to show the important codon/RNA optimization strategiescould boost the expression levels/adjuvant effects of a designedsynthetic IL-12.

293T cells (7.5×10⁵) were transfected in 6-well plates with 2 or 4 μg ofhuIL12-opt or huIL12-nonopt, respectively, using FuGene6 TransfectionReagent (Roche Applied Science, Indianapolis, Ind.) per manufacturer'sinstructions. DNA and FuGene6 Transfection Reagent were added insequence to serum-free media at a DNA:FuGene6 ratio of 1 μg DNA:3 μlFuGene6 reagent. The volume of serum-free media was determined by theamount needed to make the entire mixture's total volume equal 200 μl.The mixture was added to each well of cells and incubated for 48 hoursat 37° C. in a 5% C02 environment. At the end of the incubation, thesupernatant samples were collected for the ELISA assay.

High protein binding plates (Nunc, Rochester, N.Y.) were coated with 100μl/well of monoclonal antibody MT86/221 from the human IL-12 ELISA kit(Mabtech, Mariemont, Ohio) and incubated overnight at 4° C. After theincubation, the plates were washed twice with PBST (DPBS with 0.1% Tween20) and blocked for 1 hour with 200 μl/well of a DPBS solutionsupplemented with 0.05% Tween 20 and 0.1% BSA. Plates were subsequentlywashed with PBST. Using manufacturer's instructions, a positive standardwas prepared using hIL-12 p70 (Mabtech, Mariemont, Ohio). The positivestandard and supernatant samples were added to duplicate wells involumes of 100 μl/well at dilutions of 1:50, 1:150, 1:450, 1:1350, and1:4050. The samples and positive standard were diluted using the aboveblocking solution. The plates were subsequently incubated at 4° C.overnight. Afterwards, the plates were washed with PBST and incubatedwith 100 μl/well of mAB MT618-biotin (Mabtech, Mariemont, Ohio) for 1hour. After incubation, the plates were washed again and incubated for 1hour with 100 μl/well of Streptavidin-HRP diluted at 1:1000 in blockingbuffer. The plates were then washed again with PBST and developed usingTMB and 2N H₂S0₄. Plates were read at 450 nm using a photospectrometer.

As shown in FIGS. 1A and 1B, the huIL12-opt plasmid exhibits higherlevels of expression of IL-12 compared to the huIL12-nonopt. Clearly,the codon/RNA optimization strategies improve the expression of IL-12.

Example 2 Enhanced PSA and PSMA-Specific Cellular Immune ResponsesElicted by Vaccination with pMacIL12-opt

Rhesus macaques were immunized with 1 mg of PSA and PSMA in combinationwith 0.04 mg of pMacIL-12-opt intramuscularly followed byelectroporation with the Cellectra device from Inovio Pharmaceuticals.Two weeks after each immunization rhesus macaques were bled and PBMCswere isolated for the PSA and PSMA-specific IFN-γ ELISpot assay. Thegroup of animals receiving the pMacIL12-opt showed about 3-fold increasein peak response compared to the group of animals not receivingpMacIL12-opt (FIG. 2).

Example 3 Enhanced HBV Core and Surface Antigen-Specific Cellular ImmuneResponses Elicted by Vaccination with pMacIL12-Opt

Rhesus macaques were immunized with 1 mg of core and surface antigens incombination with 0.04 mg of pMacIL-12-opt intramuscularly followed byelectroporation with the Cellectra device from Inovio Pharmaceuticals.Two weeks after each immunization rhesus macaques were bled and PBMCswere isolated for the core and surface antigen-specific IFN-γ ELISpotassay. The group of animals receiving the pMacIL12-opt showed increasedmagnitude and breadth of cellular responses compared to the group ofanimals not receiving pMacIL12-opt (FIG. 3).

1. A composition that comprises a) a nucleic acid sequence that encodesIL-12 p35 subunit or a functional fragment thereof and b) a nucleic acidsequence that encodes IL-12 p40 subunit or a functional fragmentthereof, wherein the nucleic acid sequence that encodes IL-12 p35subunit or a functional fragment thereof is at least 98% homologous toSEQ ID NO:1 and encodes a protein at least 98% homologous to SEQ IDNO:2, or encodes a functional fragment of a nucleic acid sequence thatis at least 98% homologous to SEQ ID NO:1 and encodes a protein at least98% homologous to a functional fragment of SEQ ID NO:2; and the nucleicacid sequence that encodes IL-12 p40 subunit or a functional fragmentthereof is at least 98% homologous to SEQ ID NO:3 and encodes a proteinat least 98% homologous to SEQ ID NO:4, or encodes a functional fragmentof a nucleic acid sequence that is at least 98% homologous to SEQ IDNO:3 and encodes a protein at least 98% homologous to a functionalfragment of SEQ ID NO:4.
 2. The composition of claim 1 comprising thenucleic acid sequence that encodes IL-12 p35 subunit is at least 98%homologous to SEQ ID NO:1 and encodes a protein at least 98% homologousto SEQ ID NO:2, and the nucleic acid sequence that encodes IL-12 p40subunit is at least 98% homologous to SEQ ID NO:3 and encodes a proteinat least 98% homologous to SEQ ID NO:4.
 3. The composition of claim 2comprising the nucleic acid sequence that encodes IL-12 p35 subunit isat least 98% homologous to SEQ ID NO:1 and encodes a protein at least99% homologous to SEQ ID NO:2, and the nucleic acid sequence thatencodes IL-12 p40 subunit is at least 98% homologous to SEQ ID NO:3 andencodes a protein at least 99% homologous to SEQ ID NO:4.
 4. Thecomposition of claim 3 comprising the nucleic acid sequence that encodesIL-12 p35 subunit is at least 98% homologous to SEQ ID NO:1 and encodesSEQ ID NO:2, and the nucleic acid sequence that encodes IL-12 p40subunit is at least 98% homologous to SEQ ID NO:3 and encodes a SEQ IDNO:4.
 5. The composition of claim 2 comprising the nucleic acid sequencethat encodes IL-12 p35 subunit is at least 99% homologous to SEQ ID NO:1and encodes a protein at least 99% homologous to SEQ ID NO:2, and thenucleic acid sequence that encodes IL-12 p40 subunit is at least 99%homologous to SEQ ID NO:3 and encodes a protein at least 99% homologousto SEQ ID NO:4.
 6. The composition of claim 2 comprising the nucleicacid sequence that encodes IL-12 p35 subunit is at least 99% homologousto SEQ ID NO:1 and encodes SEQ ID NO:2, and the nucleic acid sequencethat encodes IL-12 p40 subunit is at least 99% homologous to SEQ ID NO:3and encodes a SEQ ID NO:4.
 7. The composition of claim 2 comprising thenucleic acid sequence that encodes IL-12 p35 subunit is SEQ ID NO:1, andthe nucleic acid sequence that encodes IL-12 p40 subunit is SEQ ID NO:3.8. The composition of claim 2 formulated for delivery to an individualusing electroporation.
 9. The composition of claim 2 wherein the nucleicacid sequence that encodes IL-12 p35 subunit is on a different nucleicacid molecule than the nucleic acid sequence that encodes IL-12 p40subunit.
 10. The composition of claim 2 wherein the nucleic acidsequence that encodes IL-12 p35 subunit is on a plasmid and the nucleicacid sequence that encodes IL-12 p40 subunit is on a different plasmid.11. The composition of claim 2 wherein the nucleic acid sequence thatencodes IL-12 p35 subunit and the nucleic acid sequence that encodesIL-12 p40 subunit are on the same nucleic acid molecule.
 12. Thecomposition of claim 2 wherein the nucleic acid sequence that encodesIL-12 p35 subunit and the nucleic acid sequence that encodes IL-12 p40subunit are on the same plasmid.
 13. The composition of claim 2 whereinthe nucleic acid sequence that encodes IL-12 p35 subunit and the nucleicacid sequence that encodes IL-12 p40 subunit are on the same nucleicacid molecule and operably linked to different promoters.
 14. Thecomposition of claim 2 wherein the nucleic acid sequence that encodesIL-12 p35 subunit and the nucleic acid sequence that encodes IL-12 p40subunit are on the same plasmid and operably linked to differentpromoters.
 15. The composition of claim 2 further comprising a nucleicacid sequence that encodes an immunogen.
 16. The composition of claim 2further comprising a nucleic acid sequence that encodes an immunogenfrom a pathogen selected from the group consisting of: HIV, HPV, HCV,Influenza, Smallpox, Chikungunya, foot and mouth disease virus, Malaria,human cytomegalovirus, human respiratory syncytial virus, and MRSA. 17.The composition of claim 2 wherein the nucleic acid sequence thatencodes IL-12 p35 subunit and the nucleic acid sequence that encodesIL-12 p40 subunit are incorporated into a viral particle.
 18. Thecomposition of claim 2 further comprising a nucleic acid sequence thatencodes one or more proteins selected from the group consisting of:IL-15 and IL-28. 19-24. (canceled)
 25. The composition of claim 1comprising a) the nucleic acid sequence that encodes the IL-12 p35subunit or a functional fragment thereof that is free of the codingsequence of IL-12 p35 subunit signal peptide and is optionally linked anucleic acid sequence that encodes a non-IL12 p35 signal peptide and/orb) the nucleic acid sequence that encodes the IL-12 p40 subunit or afunctional fragment that is free of the coding sequence of IL-12 p40subunit signal peptide and is optionally linked a nucleic acid sequencethat encodes a non-IL12 p40 signal peptide.
 26. The composition of claim25 comprising a) the nucleic acid sequence that encodes the IL-12 p35subunit or a functional fragment thereof that is free of the codingsequence of IL-12 p35 subunit signal peptide and linked a nucleic acidsequence that encodes SEQ ID NO:5 and/or b) the nucleic acid sequencethat encodes the IL-12 p35 subunit or a functional fragment that is freeof the coding sequence of IL-12 p40 subunit signal peptide and linked anucleic acid sequence that encodes SEQ ID NO:5.
 27. The composition ofclaim 1 comprising a) the nucleic acid sequence that encodes the IL-12p35 subunit or a functional fragment thereof that encodes amino acids23-219 of SEQ ID NO:2 and/or b) the nucleic acid sequence that encodesthe IL-12 p40 subunit or a functional fragment thereof that encodesamino acids 23-328 of SEQ ID NO:4. 28-41. (canceled)
 42. A method ofinducing an immune response against an immunogen comprisingadministering to an individual, a composition of claim 1 in combinationwith a nucleic acid sequence that encodes an immunogen in an amounteffective to induce an immune response in said individual.
 43. Themethod of claim 42 wherein the composition of claim 1 further comprisinga nucleic acid sequence that encodes an immunogen. 44-66. (canceled)